MicroRNA-34a-5p: A pivotal therapeutic target in gallbladder cancer.

Gallbladder cancer incidence has been increasing globally, and it remains challenging to expect long prognosis with the current systemic chemotherapy. We identified a novel nucleic acid-mediated therapeutic target against gallbladder cancer by using innovative organoid-based gallbladder cancer models generated from KrasLSL-G12D/+; Trp53f/f mice. Using comprehensive microRNA expression analyses and a bioinformatics approach, we identified significant microRNA-34a-5p downregulation in both murine gallbladder cancer organoids and resected human gallbladder cancer specimens. In three different human gallbladder cancer cell lines, forced microRNA-34a-5p expression inhibited cell proliferation and induced cell-cycle arrest at the G1 phase by suppressing direct target (CDK6) expression. Furthermore, comprehensive RNA sequencing revealed the significant enrichment of gene sets related to the cell-cycle regulators after microRNA-34a-5p expression in gallbladder cancer cells. In a murine xenograft model, locally injected microRNA-34a-5p mimics significantly inhibited gallbladder cancer progression and downregulated CDK6 expression. These results provide a rationale for promising therapeutics against gallbladder cancer by microRNA-34a-5p injection, as well as a strategy to explore therapeutic targets against cancers using organoid-based models, especially for those lacking useful genetically engineered murine models, such as gallbladder cancer.

Concept and Diagnostic Challenges of Renal-Limited Hemophagocytic Syndrome/Macrophage Activation Syndrome.

Hemophagocytic syndrome/macrophage activation syndrome (HPS/MAS) is a serious clinical condition that frequently leads to multiple organ failure, including acute kidney injury (AKI). Although the pathogenesis of AKI is not yet fully understood, it is believed to result from uncontrolled activation of the immune system involving macrophages and cytotoxic lymphocytes. Renal histology in HPS/MAS often presents with characteristic foamy glomerular lesions (glomerular lipidosis) with massive macrophage infiltration, known as histiocytic glomerulopathy. In this review, we introduce the recently proposed concept of renal-limited HPS/MAS as a novel etiology of histiocytic glomerular lipidosis. Patients with renal-limited HPS/MAS often develop AKI but do not fulfill the diagnostic criteria for HPS/MAS because their systemic manifestations are less severe. Therefore, the diagnosis largely depends on characteristic histological findings, that is, diffuse and global glomerular accumulation of foamy macrophages and cytotoxic lymphocytes accompanied by the interaction of these cells as well as the exclusion of various differential diseases. Although there are no established therapeutic regimens, these patients receive various types of therapies, including high-dose glucocorticoids, immunosuppressants, or anti-interleukin-1 drug, and generally achieve favorable outcomes. We summarized the concept, diagnostic challenges, and recent topics of this disease entity and discussed treatment options based on our own experiences.

Severe Type 2 Inflammation Leads to High Platelet-Activating-Factor-Associated Pathology in Chronic Rhinosinusitis with Nasal Polyps-A Hierarchical Cluster Analysis Using Bulk RNA Barcoding and Sequencing.

Platelet-activating factor (PAF) is a phospholipid-derived inflammatory mediator that triggers various inflammatory conditions, including eosinophil activation and recruitment. This study aimed to evaluate the expressions of PAF-metabolism-associated genes, namely genes coding the enzymes involved in PAF synthesis (LPCAT1, LPCAT2, LPCAT3, and LPCAT4), PAF degradation (PAFAH1B2, PAFAH1B3, and PAFAH2), and the gene for the PAF receptor (PTAFR) in subtypes of CRSwNP classified by clinical- or hierarchal-analysis-based classifications. Transcriptomic analysis using bulk RNA barcoding and sequencing (BRB-seq) was performed with CRSwNP, including eosinophilic CRS (ECRS) (n = 9), nonECRS (n = 8), ECRS with aspirin-exacerbated respiratory disease (Asp) (n = 3), and controls with a normal uncinate process mucosa (n = 6). PTAFR was only upregulated in ECRS and nonECRS. In the hierarchical cluster analysis with clusters 1 and 2 reflecting patients with low-to-moderate and high levels of type 2 inflammation, respectively, cluster 1 exhibited a significant downregulation of LPCAT2 and an upregulation of PTAFR expression, while cluster 2 showed an upregulation of LPCAT1, PAFAH1B2, and PTAFR and downregulation of PAFAH2 expression. Understanding this strong PAF-associated pathophysiology in the severe type 2 inflammation group could provide valuable insights into the treatment and management of CRSwNP.

Evaluating the State of Glomerular Disease by Analyzing Urinary Sediments: mRNA Levels and Immunofluorescence Staining for Various Markers.

Renal biopsy is the gold standard for making the final diagnosis and for predicting the progression of renal disease, but monitoring disease status by performing biopsies repeatedly is impossible because it is an invasive procedure. Urine tests are non-invasive and may reflect the general condition of the whole kidney better than renal biopsy results. We therefore investigated the diagnostic value of extensive urinary sediment analysis by immunofluorescence staining for markers expressed on kidney-derived cells (cytokeratin: marker for tubular epithelial cells, synaptopodin: marker for podocytes, claudin1: marker for parietal epithelial cells, CD68: marker for macrophages (MΦ), neutrophil elastase: marker for neutrophils). We further examined the expression levels of the mRNAs for these markers by real-time reverse transcription polymerase chain reaction. We also examined the levels of mRNAs associated with the M1 (iNOS, IL-6) and M2 (CD163, CD204, CD206, IL-10) MΦ phenotypes. Evaluated markers were compared with clinical and histological findings for the assessment of renal diseases. Claudin1- and CD68-positive cell counts in urinary sediments were higher in patients with glomerular crescents (especially cellular crescents) than in patients without crescents. The relative levels of mRNA for CD68 and the M2 MΦ markers (CD163, CD204, CD206, and IL-10) in urinary sediments were also higher in patients with glomerular crescents. These data suggest that immunofluorescence staining for claudin1 and CD68 in urinary sediments and the relative levels of mRNA for CD68 and M2 MΦ markers in urinary sediments are useful for evaluating the state of glomerular diseases.

Anti-factor H antibody-positive C3 glomerulonephritis secondary to poststreptococcal acute glomerulonephritis with diabetic nephropathy.

Poststreptococcal acute kidney glomerulonephritis (PSAGN) has been seen in adults in recent years, especially in patients with type 2 diabetes mellitus, and the renal prognosis has not always been good. There have been cases of PSAGN in which complete remission was not achieved and hematuria and proteinuria persisted, leading to end-stage renal disease. Previous reports showed that the patients subjected to PSAGN have an underlying defect in regulating the alternative pathway of complement, and they identified that antibodies to the C3 convertase, C3 nephritic factors (C3NeF), are involved. C3NeF stabilizes C3 convertase, sustains C3 activation, and causes C3 glomerulonephritis (C3GN). On the other hand, factor H is a glycoprotein that suppresses the overactivation of the alternative pathway by decaying the C3 convertase. Anti-factor H (aFH) antibodies interfere with factor H and cause the same activation of the alternative pathway as C3NeF. However, a limited number of reports describe the clinical course of C3GN with aFH antibodies. We encountered a 49-year-old Japanese man with type 2 diabetes mellitus. He was referred to our hospital because of his elevated serum creatinine, proteinuria, hematuria, and developed edema on both legs. He was diagnosed as PSAGN at the first kidney biopsy, and his renal function improved and edema and hematuria disappeared, but proteinuria persisted after 5 months. He was diagnosed as C3GN at the second kidney biopsy. In our case, no C3NeF was detected. However, a high titer of aFH antibodies was detected in stored serum from the initial presentation, providing a unified diagnosis of aFH antibody-positive C3GN secondary to PSAGN. He progressed to end-stage renal disease (ESRD) and hemodialysis was started. The persistence of high levels of aFH autoantibodies may have caused C3GN secondary to PSAGN due to activating the alternative complement pathway, which eventually worsened the nephropathy and led to ESRD.

MicroRNA-451a inhibits gemcitabine-refractory biliary tract cancer progression by suppressing the MIF-mediated PI3K/AKT pathway.

Gemcitabine is an effective chemotherapeutic agent for biliary tract cancers (BTCs), including gallbladder cancer (GBC) and cholangiocarcinoma (CCA). However, few other effective agents are currently available, particularly for GEM-refractory BTCs. We previously identified microRNA-451a (miR-451a) as a potential therapeutic target in GBC. To elucidate the antineoplastic effects of miR-451a and its underlying mechanisms, we transfected miR-451a into GBC, gemcitabine-resistant GBC (GR-GBC), and gemcitabine-resistant CCA (GR-CCA) cell lines. Furthermore, mimicking in vivo conditions, tumorigenic GBC organoids and three-dimensional (3D) cell culture systems were employed to investigate the anti-proliferative effects of miR-451a on BTCs, and its effect on stem cell properties. We found that miR-451a significantly inhibited cell proliferation, induced apoptosis, and reduced chemoresistant phenotypes, such as epithelial-mesenchymal transition, in both GBC and GR-GBC. The principal mechanism is probably the negative regulation of the phosphatidylinositol 3-kinase/AKT pathway, partially accomplished by directly downregulating macrophage migration inhibitory factor. The Gene Expression Omnibus database revealed that miR-451a was the most significantly downregulated microRNA in CCA tissues. The introduction of miR-451a resulted in similar antineoplastic effects in GR-CCA. Furthermore, miR-451a reduced cell viability in 3D spheroid models and tumorigenic GBC organoids. These findings suggest that the supplementation of miR-451a is a potential treatment strategy for GEM-refractory BTCs.

Cysts or necrotic components in pancreatic ductal adenocarcinoma is associated with the risk of EUS-FNA/B complications including needle tract seeding.

BACKGROUND: EUS-FNA/B for pancreatic ductal adenocarcinoma (PDAC) is generally considered to be safe; however, while the incidence is low, there are occurrences of complications. Among these complications, there are serious ones like needle tract seeding (NTS), and it is not known than which types of tumors have the risks of EUS-FNA/B complications. This study aimed to evaluate the risk of EUS-FNA/B complications in patients with PDAC, focusing on morphological features. METHODS: Overall, 442 patients who underwent EUS-FNA/B for solid pancreatic masses between January 2018 and May 2022 in four institutions were retrospectively surveyed. Finally, 361 patients histopathologically diagnosed with PDAC were analyzed. Among these patients, 79 tumors with cysts or necrotic components were compared with 282 tumors without cysts or necrotic components. The incidence and risk of EUS-FNA/B complications including NTS were evaluated. RESULTS: There were 9 (2.4 %) of total EUS-FNA/B complications and 3 (0.8 %) of NTS. The incidence of total complication rate and NTS in tumors with cysts or necrotic components were significantly higher than in those without cysts or necrotic components (total complication 6.3 % vs. 1.4 %, p = 0.026, NTS 3.7 % vs. 0 %, p = 0.01). The transgastric route of puncture (OR: 93.3, 95 % CI: 3.81-2284.23) and the existence of cysts or necrotic components (OR: 7.3, 95 % CI: 1.47-36.19) were risk factors for EUS-FNA/B complications identified by the multivariate analysis. CONCLUSIONS: We should pay attention to the risks of EUS-FNA/B complications, including NTS, when the tumor has cysts or necrotic components.

Acute exacerbation of immunoglobulin A nephropathy complicated by alveolar hemorrhage after coronavirus disease 2019 vaccination: A case report.

RATIONALE: Reports have suggested a relationship between coronavirus disease 2019 (COVID-19) vaccination and new-onset or recurring renal diseases, of which immunoglobulin A (IgA) nephropathy is a representative disease. Alveolar hemorrhage in patients with IgA nephropathy is rare but reportedly has a high mortality and morbidity. To our knowledge, there have been no reports regarding the development of IgA nephropathy with alveolar hemorrhage following COVID-19 vaccination. PATIENTS CONCERN: A 23-year-old Japanese man presented with hemoptysis and peripheral edema a few days after receiving a second dose of a COVID-19 mRNA vaccine. Severe renal failure and alveolar hemorrhage were noted thereafter, and renal biopsy showed crescentic glomerulonephritis with mesangial proliferation accompanied by mesangial electron-dense deposits containing IgA. Renal biopsy tissue also showed chronic histological changes suggestive of acute exacerbation of preexisting IgA nephropathy. DIAGNOSIS: The diagnosis of IgA nephropathy complicated by alveolar hemorrhage was made. INTERVENTIONS AND OUTCOMES: Renal function did not recover despite treatment with high-dose steroids; the patient was maintained on hemodialysis and eventually underwent successful renal transplantation. LESSONS: The present case suggested that although extremely rare, severe renal failure requiring renal replacement therapy could occur in patients with IgA nephropathy after COVID-19 vaccination. Future accumulation of similar cases is needed to predict the risk of renal injury following vaccination.

Glomerular lipidosis as a feature of renal-limited macrophage activation syndrome in a transplanted kidney: a case report.

BACKGROUND: Glomerular lipidosis is a rare histological feature presenting the extensive glomerular accumulation of lipids with or without histiocytic infiltration, which develops under various conditions. Among its various etiologies, macrophage activation syndrome (MAS) is a condition reported to be associated with histiocytic glomerular lipidosis. Here we describe the first case of glomerular lipidosis observed in a renal allograft that histologically mimicked histiocytic glomerulopathy owing to MAS. CASE PRESENTATION: A 42-year-old man underwent successful living-donor kidney transplantation. However, middle-grade proteinuria and increased serum triglyceride levels indicative of type V hyperlipidemia developed rapidly thereafter. An allograft biopsy performed 6 months after the transplantation showed extensive glomerular infiltration of CD68+ foam cells (histiocytes) intermingled with many CD3+ T-cells (predominantly CD8+ cells). Furthermore, frequent contact between glomerular T-cells and histiocytes, and the existence of activated CD8+ cells (CD8+, HLA-DR+ cells) were observed by double immunostaining. There was no clinicopathological data suggesting lipoprotein glomerulopathy or lecithin cholesterol acyltransferase deficiency, both of which are well-known causes of glomerular lipidosis. The histological findings were relatively similar to those of histiocytic glomerulopathy caused by MAS. As systemic manifestations of MAS, such as fever, pancytopenia, coagulation abnormalities, hyperferritinemia, increased liver enzyme levels, hepatosplenomegaly, and lymphadenopathy were minimal, this patient was clinicopathologically diagnosed as having renal-limited MAS. Although optimal treatment strategies for MAS in kidney transplant patients remains unclear, we strengthened lipid-lowering therapy using pemafibrate, without modifying the amount of immunosuppressants. Serum triglyceride levels were normalized with this treatment; however, the patient's extensive proteinuria and renal dysfunction did not improve. Biopsy analysis at 1 year after the transplantation demonstrated the disappearance of glomerular foamy changes, but the number of glomerular infiltrating cells remained similar. CONCLUSION: To our knowledge, this is the first reported case of glomerular lipidosis in a transplanted kidney. Increased interaction-activation of histiocytes (macrophages) and CD8+ T-cells, the key pathogenic feature of MAS, was observed in the glomeruli of this patient, who did not demonstrate overt systemic manifestations, suggesting a pathological condition of renal-limited MAS. The clinical effects of triglyceride-lowering therapy were limited, suggesting that hypertriglyceridemia was not the cause of but rather may be a consequence of renal-limited MAS.

Optimal liver drainage rate for survival in patients with unresectable malignant hilar biliary obstruction using 3D-image volume analyzer.

Background: Drainage exceeding 50% of total liver volume is a beneficial prognostic factor in patients with unresectable malignant hilar biliary obstruction (UMHBO). However, it is unclear what threshold percentage of total liver volume drained ('liver drainage rate') significantly improves survival in patients with UMHBO who received systemic chemotherapy. Objectives: We aimed to assess the optimal liver drainage rate that improves survival in patients with UMHBO receiving chemotherapy using a three-dimensional (3D)-image volume analyzer. Design: This study was a single-center retrospective cohort study. Methods: Data from 90 patients with UMHBO who received chemotherapy after endoscopic biliary drainage using metal stents at Okayama University Hospital from January 2003 to December 2020 were reviewed. The liver drainage rate was calculated by dividing the drained liver volume by the total liver volume using a 3D-image volume analyzer. The primary endpoint was overall survival by liver drainage rate. The secondary endpoints were time to recurrent biliary obstruction (TRBO) and prognostic factors. Results: The median total liver volume was 1172 (range: 673-2032) mL, and the median liver drainage rate was 83% (range: 50-100). Overall survival was 376 (95% CI: 271-450) days, and patients with >80% drainage (n = 67) had significantly longer survival than those with <80% drainage (n = 23) (450 days versus 224 days, p = 0.0033, log-rank test). TRBO was 201 (95% CI: 155-327) days and did not differ significantly by liver drainage rate. Multivariate Cox proportional hazards regression analysis revealed >80% liver drainage [hazard ratio (HR): 0.35, 95% CI: 0.20-0.62, p = 0.0003] and hilar cholangiocarcinoma (HR: 0.30, 95% CI: 0.17-0.50, p < 0.0001) as significant prognostic factors. Conclusion: In patients with UMHBO scheduled for chemotherapy, >80% drainage is associated with improved survival. Further prospective multicenter studies are needed to verify the results of this study. Trail registration: Okayama University Hospital, IRB number: 2108-011.

Exhaled Nitric Oxide and Olfactory Dysfunction in Patients with Asthma: Association with Chronic Rhinosinusitis.

Objectives: Olfactory dysfunction is a clinical sign that is important to detect with coexistent upper airway comorbidities in patients with asthma. This study aimed to investigate the etiology of olfactory dysfunction in patients with asthma and the relationship between fractional exhaled nitric oxide (FeNO) levels. Materials and Methods: This study included 47 asthma patients who were evaluated for olfactory dysfunction at Hiroshima University Hospital between 2012 and 2020. The etiologies of olfactory dysfunction were evaluated, and they were classified according to the FeNO levels of patients with asthma. Results: Olfactory dysfunction was observed in 30 patients with asthma, with chronic rhinosinusitis (77%) being the most prevalent etiology. Eosinophilic chronic rhinosinusitis (ECRS) was the most prevalent etiology of olfactory dysfunction in asthma patients with high FeNO levels (≥25 ppb), while non-eosinophilic chronic rhinosinusitis (NCRS) was the most prevalent etiology in asthma patients with low FeNO levels (<25 ppb). Additionally, the prevalence of ECRS was significantly higher in asthma patients with olfactory dysfunction and high FeNO levels (74%) than in those with either high FeNO levels or olfactory dysfunction and those with low FeNO levels and no olfactory dysfunction (12% and 9%, respectively). Conclusions: We found that ECRS was the predominant cause of olfactory dysfunction in patients with high FeNO levels, while NCRS was more common in those with low FeNO levels. The present study showed that both ECRS and NCRS are common etiologies of olfactory dysfunction in patients with asthma. Additionally, this study supports the link between upper and lower airway inflammation in patients with asthma complicated with olfactory dysfunction.

Histological transition from minimal change disease to THSD7A-associated membranous nephropathy in a patient receiving long-term steroid treatment: A case report.

RATIONALE: A predominant Th2 immune response is suggested in the pathogenesis of both minimal change disease (MCD) and membranous nephropathy (MN); however, consecutive development of the 2 diseases in a patient is extremely rare. PATIENT CONCERN: A Japanese man, who developed nephrotic syndrome in his 50s and was diagnosed with MCD by renal biopsy, experienced a relapse of proteinuria approximately 3 years later during long-term steroid treatment. Since the proteinuria was resistant to increase in steroid dosage, repeat renal biopsy was performed, which revealed a small amount of glomerular subepithelial immune deposits containing immunoglobulin (Ig)G (dominantly IgG4). Immunostaining for thrombospondin-type-1-domain-containing-7A (THSD7A) was positive on the glomerular capillary walls, whereas that for other causative antigens of MN, such as phospholipase A2 receptor or neural epidermal growth factor-like 1 protein, was negative. Detailed examination found no associated condition, including malignancies and allergic diseases. DIAGNOSIS: The diagnosis of THSD7A-associated idiopathic MN was made. INTERVENTIONS AND OUTCOMES: He received further increased dose of steroids. Thereafter he maintained clinical improvement because his urinary protein level was decreased. LESSONS: The present case suggested that histological transition from MCD to MN is possible and repeat biopsy would be crucial for accurate diagnosis.

Time-resolved study on signaling pathway of photoactivated adenylate cyclase and its nonlinear optical response.

Photoactivated adenylate cyclases (PACs) are multidomain BLUF proteins that regulate the cellular levels of cAMP in a light-dependent manner. The signaling route and dynamics of PAC from Oscillatoria acuminata (OaPAC), which consists of a light sensor BLUF domain, an adenylate cyclase domain, and a connector helix (α3-helix), were studied by detecting conformational changes in the protein moiety. Although circular dichroism and small-angle X-ray scattering measurements did not show significant changes upon light illumination, the transient grating method successfully detected light-induced changes in the diffusion coefficient (diffusion-sensitive conformational change (DSCC)) of full-length OaPAC and the BLUF domain with the α3-helix. DSCC of full-length OaPAC was observed only when both protomers in a dimer were photoconverted. This light intensity dependence suggests that OaPAC is a cyclase with a nonlinear light intensity response. The enzymatic activity indeed nonlinearly depends on light intensity, that is, OaPAC is activated under strong light conditions. It was also found that both DSCC and enzymatic activity were suppressed by a mutation in the W90 residue, indicating the importance of the highly conserved Trp in many BLUF domains for the function. Based on these findings, a reaction scheme was proposed together with the reaction dynamics.

Involvement of Innate Immune System in the Pathogenesis of Sepsis-Associated Acute Kidney Injury.

Although experimental models have shown that the innate immune system is a main contributor to acute kidney injury (AKI), its involvement in human sepsis-associated AKI (SA-AKI) remains unclear. We retrospectively evaluated 19 patients with SA-AKI who were treated with continuous renal replacement therapy (CRRT). Serum cytokine, complement components, and the proportion and functions of innate immune cells, such as CD56+ T cells, CD56+ natural killer (NK) cells, and monocytes, were analyzed. There were no differences in the proportions of CD56+ T and NK cells between patients with SA-AKI and healthy controls. In patients with SA-AKI, fas ligand (FasL) expression in CD56+ T cells was significantly upregulated, and the proportion of perforin-positive CD56+ T cells tended to be higher than that in healthy controls. The positive rate of both FasL and perforin of CD56+ T cells was significantly higher than that of CD56- T cells, which include cytotoxic T cells. Antigen-presenting capacity and phagocytic activity of monocytes in patients with SA-AKI were significantly decreased compared to those of healthy controls and did not recover soon after the initiation of CRRT. CD56+ T cells are involved in the disease processes of human SA-AKI through effector molecules such as FasL or perforin.

Infection-Related Cryoglobulinemic Glomerulonephritis with Serum Anti-Factor B Antibodies Identified and Staining for NAPlr/Plasmin Activity Due to Infective Endocarditis.

In this rare case of infection-related cryoglobulinemic glomerulonephritis with infective endocarditis, a 78-year-old male presented with an acute onset of fever and rapidly progressive glomerulonephritis. His blood culture results were positive for Cutibacterium modestum, and transesophageal echocardiography showed vegetation. He was diagnosed with endocarditis. His serum immunoglobulin M, IgM-cryoglobulin, and proteinase-3-anti-neutrophil cytoplasmic antibody levels were elevated, and his serum complement 3 (C3) and C4 levels were decreased. Renal biopsy results showed endocapillary proliferation, mesangial cell proliferation, and no necrotizing lesions on light microscopy, with strong positive staining for IgM, C3, and C1q in the capillary wall. Electron microscopy showed deposits in the mesangial area in the form of fibrous structures without any humps. Histological examination confirmed a diagnosis of cryoglobulinemic glomerulonephritis. Further examination showed the presence of serum anti-factor B antibodies and positive staining for nephritis-associated plasmin receptor and plasmin activity in the glomeruli, suggesting infective endocarditis-induced cryoglobulinemic glomerulonephritis.

Dynamic Solution Structures of Whole Human NAP1 Dimer Bound to One and Two Histone H2A-H2B Heterodimers Obtained by Integrative Methods.

Nucleosome assembly protein 1 (NAP1) binds to histone H2A-H2B heterodimers, mediating their deposition on and eviction from the nucleosome. Human NAP1 (hNAP1) consists of a dimerization core domain and intrinsically disordered C-terminal acidic domain (CTAD), both of which are essential for H2A-H2B binding. Several structures of NAP1 proteins bound to H2A-H2B exhibit binding polymorphisms of the core domain, but the distinct structural roles of the core and CTAD domains remain elusive. Here, we have examined dynamic structures of the full-length hNAP1 dimer bound to one and two H2A-H2B heterodimers by integrative methods. Nuclear magnetic resonance (NMR) spectroscopy of full-length hNAP1 showed CTAD binding to H2A-H2B. Atomic force microscopy revealed that hNAP1 forms oligomers of tandem repeated dimers; therefore, we generated a stable dimeric hNAP1 mutant exhibiting the same H2A-H2B binding affinity as wild-type hNAP1. Size exclusion chromatography (SEC), multi-angle light scattering (MALS) and small angle X-ray scattering (SAXS), followed by modelling and molecular dynamics simulations, have been used to reveal the stepwise dynamic complex structures of hNAP1 binding to one and two H2A-H2B heterodimers. The first H2A-H2B dimer binds mainly to the core domain of hNAP1, while the second H2A-H2B binds dynamically to both CTADs. Based on our findings, we present a model of the eviction of H2A-H2B from nucleosomes by NAP1.